1. Field of the Invention
The invention relates to the construction of protease-deficient bacteria, particularly E. coli, using methods of recombinant DNA technology, and to the expression of polypeptides by the protease-deficient cells. In a particular aspect of the invention, improved yields of protease sensitive polypeptide products are accumulated when a multiply protease-deficient E. coli is used to generate proteolytically sensitive proteins.
2. Description of Related Art
Escherichia coli has been the most widely used microorganism for the production of commercially important recombinant proteins. Despite the lack of certain kinds of post-translational processing and the production of endotoxins, E. coli presents numerous advantages for protein expression. Its genetics are well understood, it can be grown to high densities on inexpensive substrates, and fermentation scale-up is straightforward (1).
A number of useful eukaryotic proteins have been cloned and expressed in E. coli including human insulin and proinsulin, human and bovine somatotropins, interferons and tissue plasminogen activator. Recently, Huse and coworkers (2) constructed a bacteriophage lambda system which allows the expression and rapid screening of mouse F.sub.ab antibody fragments in E. coli. For most biotechnology applications it is advantageous to secrete the protein from the cytoplasm. Secretion of the polypeptide product can facilitate correct folding, reduce protein degradation and simplify subsequent purification steps.
One of the major problems associated with the expression of heterologous polypeptides in Escherichia coli is the degradation of cloned gene products by host-specific proteases (3). It has been shown that, as in eukaryotic cells, energy-dependent processes are important for the degradation of E. coli proteins with abnormal conformations (4,5). However, most E. coli proteases hydrolyze peptide bonds via an energy-independent pathway. At least 25 proteases and peptidases have been identified in different cellular compartments of E. coli (6,7). The biochemical characterization of these enzymes is incomplete and there is relatively little information on their physiological role. One or more of these proteases may act upon any given polypeptide to effect degradation and thereby reduce yields, sometimes quite drastically.
One approach to solving the problem of low polypeptide production in bacterial host cells has been the use of an inducible expression system in combination with a constitutively protease-deficient bacterial host strain. This method will operate to increase polypeptide yields only if the deficient protease has as its substrate the target polypeptide which is being expressed. For example, the production of an immunologically functional antibody fragment in a constitutively lon.sup.- and/or htpR.sup.- E. coli strain produced low yields (8) even though such strains have been shown to have a general defect in protein degradation.
Several strains of E. coli deficient in proteases or genes controlling the regulation of proteases are known (9-11). Some of these strains have been used in attempts to efficiently produce proteolytically sensitive polypeptides, particularly those of potential medical or other commercial interest.
Some singly protease-deficient mutants of Escherichia coli have been prepared. These include a degP deficient genetically engineered strain and a spontaneous mutant, UT4400, in which the entire ompT gene together with a sizable piece of adjacent DNA have been deleted from the chromosome (12). Mutants carrying large deletions in the Protease III (ptr) gene including adjacent genes recC, recB and recD, have been isolated (10). However, since the adjacent genes are important for cell viability and stable propagation of plasmids in Escherichia coli, the mutant strains exhibit growth defects and low protein production. A Protease III mutant strain has also been isolated after chemical mutagenesis (13).
Using a similar rationale, but a genetic engineering approach, a method of mutagenizing Escherichia coli to produce a cell with a defective periplasmic protease has been described (9). A degP deletion mutant was constructed and recombined into an E. coli chromosome (14). Some workers have shown that the proteolytically sensitive fusion protein, protein A-.beta.-lactamase, is stabilized three-fold in such a deg mutant (15,16).
Most proteins are degraded by more than one protease. Therefore, the use of mutants deficient in the synthesis of a single enzyme can only partially prevent the degradation of the product. Inactivation of multiple proteolytic enzymes may lead to higher production. The challenge is complex, however, because there is no assurance that disablement or deletion of any given protease or combination of proteases will result in a viable or unchanged host cell or that such manipulation will avoid the precipitation of toxic events within the cell.
The genetically engineered protease-deficient microorganisms of the present invention have shown unexpectedly improved yields of proteolytically sensitive polypeptide products while maintaining good growth and cell viability. For the first time, a microorganism deficient in Protease III has been engineered which, unlike mutants previously isolated, is not defective in the gene product of adjacent genes recC, recB or recD. In addition, three doubly protease defective microorganisms and a triply defective microorganism have been engineered. The use of these microorganisms grown under optimized conditions significantly broadens the potential to produce commercially valuable polypeptides.